The present invention relates to compositions and methods for inhibition of nitric oxide (NO), and to therapeutic treatment of diseases or disorders that involve inappropriate or detrimental NO activity. Thus, the invention relates to modulation of cellular activities, including macrophage activity, endothelial cell function, and the like. The present invention also relates to substances exhibiting inhibitory activity toward nitric oxide-associated diseases, which are facilitated by serine protease activity. More particularly, the inhibitory compounds comprise naturally occurring and man-made serine protease inhibitors and antagonists.
2.1. Serine Proteases
Serine proteases serve an important role in human physiology by mediating the activation of vital functions. In addition to their normal physiological function, serine proteases have been implicated in a number of pathological conditions in humans. Serine proteases are characterized by a catalytic triad consisting of aspartic acid, histidine and serine at the active site.
The naturally occurring serine protease inhibitors are usually, but not always, polypeptides and proteins which have been classified into families primarily on the basis of the disulfide bonding pattern and the sequence homology of the reactive site. Serine protease inhibitors, including the group known as serpins, have been found in microbes, in the tissues and fluids of plants, animals, insects and other organisms. Protease inhibitor activities were first discovered in human plasma by Fermi and Pemossi in 1894. At least nine separate, well-characterized proteins are now identified, which share the ability to inhibit the activity of various proteases. Several of the inhibitors have been grouped together, namely xcex11-proteinase inhibitor, antithrombin III, antichymotrypsin, C1-inhibitor, and xcex12-antiplasmin, which are directed against various serine proteases, i.e., leukocyte elastase, thrombin, cathepsin G, chymotrypsin, plasminogen activators, and plasmin. These inhibitors are members of the xcex11-proteinase inhibitor class. The protein xcex12-macroglobulin inhibits members of all four catalytic classes: serine, cysteine, aspartic, and metalloproteases. However, other types of protease inhibitors are class specific. For example, the xcex11-proteinase inhibitor (also known as (xcex11-antitrypsin or AAT) and inter-alpha-trypsin inhibitor inhibit only serine proteases, xcex11-cysteine protease inhibitor inhibits cysteine proteases, and xcex11-anticollagenase inhibits collagenolytic enzymes of the metalloenzyme class.
Human neutrophil elastase (NE) is a proteolytic enzyme secreted by polymorphonuclear leukocytes in response to a variety of inflammatory stimuli. The degradative capacity of NE, under normal circumstances, is modulated by relatively high plasma concentrations of xcex11-antitrypsin. However, stimulated neutrophils produce a burst of active oxygen metabolites, some of which (hypochlorous acid for example) are capable of oxidizing a critical methionine residue in xcex11-antitrypsin. Oxidized xcex11-antitrypsin has been shown to have a limited potency as a NE inhibitor and it has been proposed that alteration of this protease/antiprotease balance permits NE to perform its degradative functions in localized and controlled environments.
xcex11-Antitrypsin is a glycoprotein of MW 51,000 with 417 amino acids and 3 oligosaccharide side chains. Human xcex11-antitrypsin was named anti-trypsin because of its initially discovered ability to inactivate pancreatic trypsin. Human xcex11-antitrypsin is a single polypeptide chain with no internal disulfide bonds and only a single cysteine residue normally intermolecularly disulfide-linked to either cysteine or glutathione. The reactive site of xcex11-antitrypsin contains a methionine residue, which is labile to oxidation upon exposure to tobacco smoke or other oxidizing pollutants. Such oxidation reduces the biological activity of xcex11-antitrypsin; therefore substitution of another amino acid at that position, i.e. alanine, valine, glycine, phenylalanine, arginine or lysine, produces a form of xcex11-antitrypsin which is more stable. xcex11-Antitrypsin can be represented by the following formula:
Ciliberto, et al. in Cell 1985, 41, 531-540. The critical amino acid sequence near the carboxyterminal end of xcex11-antitrypsin is shown in bold and is pertinent to this invention.
The C-terminus of human xcex11-antitrypsin is homologous to antithrombin (ATIII), antichymotrypsin (ACT), C1-inhibitor, tPA-inhibitor, mouse anti-trypsin, mouse contrapsin, barley protein Z, and ovalbumin. The normal plasma concentration of ATT ranges from 1.3 to 3.5 mg/ml although it can behave as an acute phase reactant and increases 3-4-fold during host response to inflammation and/or tissue injury such as with pregnancy, acute infection, and tumors. It easily diffuses into tissue spaces and forms a 1:1 complex with a target protease, principally neutrophil elastase. Other enzymes such as trypsin, chymotrypsin, cathepsin G, plasmin, thrombin, tissue kallikrein, and factor Xa can also serve as substrates. The enzyme/inhibitor complex is then removed from circulation by binding to serpin-enzyme complex (SEC) receptor and catabolized by the liver and spleen. Humans with circulating levels of xcex11-antitrypsin less than 15% of normal are susceptible to the development of lung disease, e.g., familial emphysema, at an early age. Familial emphysema is associated with low ratios of xcex11-antitrypsin to serine proteases, particularly elastase. Therefore, it appears that this inhibitor represents an important part of the defense mechanism against attack by serine proteases.
xcex11-Antitrypsin is one of few naturally occurring mammalian serine protease inhibitors currently approved for the clinical therapy of protease imbalance. Therapeutic xcex11-antitrypsin has been commercially available since the mid 80s and is prepared by various purification methods (see for example Bollen et al., U.S. Pat. No. 4,629,567; Thompson et al., U.S. Pat. Nos. 4,760,130; 5,616,693; WO 98/56821). Prolastin is a trademark for a purified variant of xcex11-antitrypsin and is currently sold by Bayer Company (U.S. Pat. No. 5,610,285 Lebing et al., Mar. 11, 1997). Recombinant unmodified and mutant variants of xcex11-antitrypsin produced by genetic engineering methods are also known (U.S. Pat. No. 4,711,848); methods of use are also known, e.g., (xcex11-antitrypsin gene therapy/delivery (U.S. Pat. No. 5,399,346 to French Anderson et al.).
The two known cellular mechanisms of action of serine proteases are by direct degradative effects and by activation of G-protein-coupled proteinase-activated receptors (PARs). The PAR is activated by the binding of the protease followed by hydrolysis of specific peptide bonds, with the result that the new N-terminal sequences stimulate the receptor. The consequences of PAR activation depend on the PAR type that is stimulated and on the cell or tissue affected and may include activation of phospholipase Cxcex2, activation of protein kinase C and inhibition of adenylate kinase (Dery, O. and Bunnett, N. W. Biochem Soc Trans 1999, 27,246-254; Altieri, D. C. J. Leukoc Biol 1995, 58, 120-127; Dery, O. et al. Am J. Physiol 1998, 274, C1429-C1452).
2.2. Nitric Oxide (NO)
Nitric oxide (NO), also known as endothelium-derived relaxing factor (EDRF), is a potent vasodilator, oxidant, and neurotransmitter produced by many different types of cells and tissues, such as endothelium, macrophages and neuronal cells reviewed by Patel R. P., et al. in Biochim Biophys Acta 1999, 1411, 385-400; Lowenstein, C. J. and Snyder, S. H. in Cell 1992, 70, 705-707; Nathan, C. in FASEB J. 1992, 6, 3051.
A presently dominant theory based on DNA analyses holds that the NO synthase enzymes (NOS) exist in at least three isoforms, namely, neuronal constitutive NOS (N-cNOS) which is present constitutively in neurons, endothelial constitutive NOS (E-cNOS) which is present constitutively in endothelial cells, and inducible NOS (iNOS) which is expressed following stimulation by cytokines and lipopolysaccharides in macrophages and many other cells. (see Beck, K. F. et al. in J. Exp Biol 1999, 202, 645-53; Kirkeboen, K. A. and Strand, O. A. in Acta Anaesthesiol Scand 1999, 43, 275; Wood, E. R. et al. in Biochem Biophys Res Commun 1993, 191, 767-74; Lowenstein C. J. et al. in Proc. Natl. Acad. Sci. USA, 1993, 90, 9730). Among these three isoforms, N-cNOS and E-cNOS are calcium-dependent whereas iNOS is calcium-independent (Nathan, C. in FASEB J. 1992, 6, 3051). NO synthesized by nitric oxide synthase from arginine and oxygen is also an important signal transducing molecule in various cell types (Nathan, 1992, supra). In macrophages NO has assumed, under certain situations, the role of a cytotoxic agentxe2x80x94a reactive nitrogen intermediate that is lethal to cancer cells and microorganisms. The release of nitric oxide is also involved in other acute and chronic inflammatory diseases. These diseases include but are not limited to diseases such as, for example, acute and chronic infections (viral, bacterial and fungal), acute and chronic bronchitis, sinusitis, and upper respiratory infections, including the common cold; acute and chronic gastroenteritis and colitis; acute and chronic cystitis, and urethritis; acute and chronic dermatitis; acute and chronic conjunctivitis; acute and chronic serositis (pericarditis, peritonitis, synovitis, pleuritis and tendinitis); uremic pericarditis; acute and chronic cholecystitis; acute and chronic vaginitis; drug reactions; insect bites; bums and sunburn.
Released NO combines very rapidly with superoxide to form peroxynitrite (ONOOxe2x88x92xe2x80xa2), a reactive tissue damaging nitrogen species thought to be involved in the pathology of several chronic diseases. Peroxynitrite nitrates tyrosine residues and inactivates xcex11-antitrypsin (Rehman, A. et al. in Br J Pharmacol, 1997, 122, 1702). This mechanism is postulated to be responsible for xcex11-antitrypsin inactivation by cigarette smoke (Pryor, W. A. et al., in Chem Biol Interact 1985, 54, 171). Nitric oxide inhibits iron-containing enzymes important in respiration and DNA synthesis. Peroxynitrite decomposes to the reactive NO2 and hydroxyl radicals, and NO stimulates ADP-ribosylation of various proteins including glyceraldehyde-3-phosphate dehydrogenase, with consequent inactivation.
Van Molle and colleagues have shown that the acute phase protein xcex11-antitrypsin inhibits the cellular lethality induced by tumor necrosis factor (TNF) both in normal mice and in mice sensitized with galactosamine but similar apoptosis of hepatocytes induced by anti-Fas remained unaffected. Molle W. et al. in J Immunol 1997, 159, 3555. However, xcex11-antitrypsin did not affect the induction by TNF of NO Van Molle, ibid. In contrast, Bratt and colleagues have shown that TNF injury was not prevented by xcex11-antitrypsin (Bratt, J. and Palmblad, J. in J Immunol 1997, 159, 812).
Many proteins are reported to modulate NO production. Macrophage deactivating factor and TGF-xcex2 partially blocked NO release by macrophages activated with xcex3-interferon (xcex3-IFN or IFN-xcex3) and TGF-xcex1 (transforming growth factor-xcex1), but not when activated by xcex3-IFN and lipopolysaccharide (LPS or endotoxin) (Ding, A. et al., in J. Immunol. 1990, 145, 940). Epidermal growth factor can suppress both NO and H2O2 production by keratinocytes (Heck, D. E. et al., in J. Biol. Chem. 1990, 267, 21277). Incubation of LPS-activated peritoneal neutrophils with IL-8 blocks both the release of NO and NOS induction at the transcriptional level (McCall, T. B. et al., in Biochem. Biophys. Res. Commun. 1992, 186, 680).
TGF-xcex21 and 12-O-tetradecanoylphorbol-13-acetate (i.e., phorbol myristyl acetate or PMA) inhibit LPS and xcex3-IFN-induced NO synthesis in mouse bone marrow cells (Punjabi, C. J. et al., in J. Immunol. 1992, 149, 2179). In contrast, in bovine pigmented retinal epithelial cells TGF-xcex2 increases the NO production, as measured by nitrite, attributable to treatment with LPS and xcex3-IFN. In this system both fibroblast growth factor (FGF)-1 and FGF-2 inhibit nitrite production, likely by inhibiting the induction of NOS mRNA at the transcriptional level (Goureau, O. et al., in Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 4276). Insulin-like growth factor 1 reduces the amount of NO produced by the action of IL-162  on vascular smooth muscle cells (Schini et al. in Circ Res 1994, 74, 24). The fact that so many agents can modulate NO activity by increasing or inhibiting NO production suggests that NO production may be important in many different contexts.
The overproduction in the body of nitric oxide (NO) and/or peroxynitrite (ONOOxe2x88x92xe2x80xa2) has been suggested by some to be a contributing factor to diseases that are immune-mediated and/or inflammatory. In a clinical study the levels of IL-6, IL-1xcex2, NO and xcex11-antitrypsin were shown to be involved in the pathogenesis of scorpion envenomation and correlated with the severity of envenomation (Meki, A. R. et al. in Toxicon 1998, 36, 18519). An extensively used model system to study multiple sclerosis, an example of a disease treated by the present invention, is experimental allergic encephalomyelitis (EAE) in rats and mice. (Popko B. and Baerwald, K. D. in Neurochem Res 1999, 24, 331; Smith, M. E. in Neurochem Res 1999, 24, 261).
Thus, the prior art taught that NO metabolites inactivate xcex11-antitrypsin. Also taught was that in certain clinical situations NO levels tended to rise concomitantly along with increase in xcex11-antitrypsin levels, although the AAT activity may have been reduced. However, the prior art failed to recognize that xcex11-antitrypsin might in fact prevent NO synthesis. The present inventor discovered that therapeutic and physiological levels of xcex11-antitrypsin can efficiently block xcex3-IFN- and LPS-induced NO synthesis. This invention addresses a long-felt need for safe and effective amelioration of many diseases related to nitric oxide-caused damage.
The present invention is directed to a method for treating a disease or disorder involving an excess activity of nitric oxide (NO) in an animal subject. The method of the invention comprises administering a therapeutically effective amount of an agent that reduces NO levels, to an animal subject suspected of having a disease or disorder involving excess nitric oxide. In a preferred embodiment the agent can be xcex11-antitrypsin. In addition, peptides of interest are homologous and analogous peptides. While homologues are natural peptides with sequence homology, analogues will be peptidyl derivatives, e.g., aldehyde or ketone derivatives of such peptides. Typical examples of analogues are TLCK or TPCK. Without limiting to xcex11-antitrypsin and peptide derivatives of xcex11-antitrypsin, compounds like oxadiazole, thiadiazole, CE-2072, UT-77, and triazole peptoids are preferred. The agent that reduces NO levels can also be an xcex11-antitrypsin-like agent, an inhibitor of elastase, or an inhibitor of proteinase-3. The xcex11-antitrypsin-like agent can include, but is not limited to, small organic molecules including naturally-occurring, synthetic, and biosynthetic molecules, small inorganic molecules including naturally-occurring and synthetic molecules, natural products including those produced by plants and fungi, peptides, variants of xcex11-antitrypsin, chemically modified peptides, and proteins. An xcex11-antitrypsin-like agent has the capability of inhibiting the proteolytic activity of trypsin, elastase, kallikrein, and/or other serine proteases.
A general method of treating a mammal suffering from a pathological condition that is mediated by endogenous serine protease or serine protease-like activity is contemplated as well, which comprises administering a therapeutically effective amount of a substance exhibiting mammalian xcex11-antitrypsin or xcex11-antitrypsin-like activity. The pathological condition can be precipitated at least in part by abnormal nitric oxide levels.
Also a method is provided of inhibiting bacterial colonization in a host, which comprises administering to a mammal susceptible to bacterial colonization an effective amount of a substance exhibiting mammalian xcex11-antitrypsin or xcex11-antitrypsin-like activity. Without limiting to xcex11-antitrypsin, the substance may be a compound that inhibits proteinase-3, cathepsin G, or elastase.
Also contemplated is a method of preventing a deficiency of functional endogenous xcex11-antitrypsin levels in a patient susceptible to an infection that is mediated by endogenous host serine protease or serine protease-like activity, by treating with a pharmaceutical composition in a pharmaceutically acceptable carrier comprising effective amounts of a substance exhibiting mammalian xcex11-antitrypsin or xcex11-antitrypsin-like activity. In addition, to reduce ischemia-reperfusion injury associated with administration of thrombolytics, a combination of serine protease inhibitor, and a thrombolytic agent such as tissue plasminogen activator, urokinase, streptokinase, or combinations or complexes thereof can be administered. The pharmaceutical composition can be a peptide or a small molecule, which exhibits xcex11-antitrypsin or xcex11-antitrypsin-like activity.
It should be apparent that in addition to these preferred embodiments a method is contemplated which consists of treating an individual having a pathological condition caused, in whole or part, by nitric oxide release. In accordance with this embodiment, a method of inhibiting nitric oxide release is provided wherein the target of the therapy is a cell and one will contact such cell with an effective amount of a compound having xcex11-antitrypsin activity.
According to the invention, the peptide can be protected or derivitized in various ways, e.g., N-terminal acylation, C-terminal amidation, cyclization, etc. In a specific embodiment, the N-terminus of the peptide is acetylated.
The invention further provides pharmaceutical compositions comprising such agents. In yet a further embodiment of the invention, the pharmaceutical composition also comprises a vasoconstrictor effective to increase blood pressure in an animal.
It is therefore the goal of the present invention, in its broadest aspect, to provide methods of treating diseases dependent on the action of NO and proteases. Accordingly, it should be recognized that this invention is applicable to the control of catalytic activity of serine proteases in any appropriate situation including, but not necessarily limited to, medicine, biology, agriculture, and microbial fermentation.
Accordingly, it is therefore the overall object of the present invention to provide compounds that exhibit inhibitory activity toward serine proteases.
It is an object of the present invention to provide clinically acceptable serine protease inhibitors with recognized utility and exhibiting relatively high activity at relatively low concentrations.
It is yet another object of the invention to provide means of regulating nitric oxide release by compounds having xcex11-antitrypsin activity.
These and other objects and advantages of the present invention will be recognized by those skilled in the art from the following description and illustrative examples.
FIG. 1 illustrates the effect of xcex11-antitrypsin on NO release upon induction with LPS and xcex3-IFN.
FIG. 2 illustrates the effect of xcex11-antitrypsin on induction of iNOS protein by LPS and xcex3-interferon.
FIG. 3 illustrates an electrophoretic mobility shift assay of NF-xcexaB on gel electrophoresis demonstrating inhibition of NF-xcexaB activation due to the presence of xcex11-antitrypsin.
FIG. 4 illustrates the inhibition of elevated NO levels, measured as NO2xe2x88x92, by CE-2072.
FIG. 5 illustrates the inhibition of p-ERK expression by xcex11-antitrypsin (AAT).
FIG. 6 illustrates the effect of xcex11-antitrypsin on cytomegalovirus replication.
FIG. 7 illustrates the effect of xcex11-antitrypsin on herpes simplex infection.